Gene Targeting Mediated By Triple Helix Forming Oligonucleotides

我们正在开发一种基于寡核苷酸的基因靶向程序，这种寡核苷酸与双链DNA中的特定序列形成稳定的三螺旋复合物。这种方法有望成为一种简单而有效的技术，用于将DNA活性化合物输送到活细胞中染色体DNA的特定位点。应用包括基因敲除，定向基因转换和重组，也许还有基因治疗。我们已经制备了含有新型糖类似物的tfo，并确定了一种修饰格式，支持在活的哺乳动物细胞中有效靶向特定的染色体序列。在我们的开发工作中，我们制备了tfo，它与一种光活性DNA诱变剂相连，直接针对一种常被用作突变报告基因的基因序列（HPRT）。我们将其引入哺乳动物细胞，并在诱变原光激活后，分离出靶基因突变的细胞菌落。序列分析表明，突变位于基因内的靶序列。用这些TFOs处理S期细胞导致30%的细胞靶向交联，5-10%的突变频率。在静止细胞中，交联和诱变都较低。这些结果表明，哺乳动物细胞中染色体靶点的可及性是由细胞的生物学特性调节的。这一策略已扩展到其他基因的遗传分析的目标是不可能的。例如，我们最近使用生化试验表明，人类-珠蛋白基因中的一个位点可以高效地靶向。对生物活性所需的寡核苷酸化学分析表明，活性tfo含有平衡的修饰，过多或过少都会降低活性。三联体结构在多嘌呤：多嘧啶序列上是最稳定的，而这种排列中断的tfo通常不成功。我们已经合成了一种新的碱基类似物，它允许在含有多嘌呤：多嘧啶元素中C:G中断的靶序列上稳定地形成三联体。我们已经证明，tfo与这种类似物在体外形成稳定的三聚物。我们通过靶向序列转换构建了一个新的细胞系，其中标准多嘌呤：多嘧啶靶标被含有C:G中断的靶标取代。然后我们用新的TFO和标准TFO处理标准细胞系和等基因细胞系。标准TFO对具有标准靶标的细胞有效，而具有新型类似物的TFO对具有中断靶标而非标准靶标的细胞有效。这是首次证明碱基类似物的生物活性，使TFO能够克服靶序列的限制。新型标准三联体结构的分子模拟结果与生物化学和生物学数据一致。

We are developing a gene targeting program based on oligonucleotides that form stable triple helix complexes with specific sequences in duplex DNA. This approach has the promise to become a simple and efficient technology for delivering DNA reactive compounds to specific sites in chromosomal DNA in living cells. Applications include gene knockout, directed gene conversion and recombination, and, perhaps, gene therapy. We have prepared TFOs containing novel sugar analogues and have identified a modification format that supports efficient targeting of specific chrosomal sequences in living mammalian cells. In our developmental work we prepared TFOs, linked to a photoactive DNA mutagen, directed against a sequence in a gene (HPRT) frequently used as a mutation reporter. We introduced this into mammalian cells and, after photoactivation of the mutagen, isolated colonies of cells with mutations in the target gene. Sequence analysis showed that the mutations were located at the target sequence within the gene. Treatment of S phase cells with these TFOs resulted in targeted crosslinking in 30% of the cells and 5-10% mutation frequencies. Both crosslinking and mutagenesis were much lower in quiescent cells. These results indicate that the accessibility of chromosomal target sites in mammalian cells is modulated by the biology of the cell. This strategy has been extended to other genes for which genetic analyses of targeting are not possible. For example, we have recently shown, using a biochemical assay, that a site in the human beta globin gene can be targeted at high efficiency. Additional analyses of the oligonucleotide chemistry required for bioactivity revealed that the active TFOs contain a balance of modifications, too much or too little reduces activity. Triplex formation is most stable on polypurine:polypyrimidine sequences, and TFOs against targets with interruptions in this arrangement are generally not successful. We have synthesized a novel base analogue that permits stable triplex formation on a target sequence containing a C:G interruption in the polypurine:polypyrimidine element. We have shown that TFOs with this analogue form stable triplexes in vitro. We constructed a novel cell line by targeted sequence conversion in which the standard polypurine:polypyrimidine target was replaced with a target containing the C:G interruption. We then treated the standard line and the isogenic line cell line with the new TFO and the standard TFO. The standard TFO was effective against the cells with the standard target while the TFO with the novel analogue was active against the cells with the interrupted target but not the standard target. This is the first demonstration of biological activity of a base analogue that enables a TFO to overcome the limitations on target sequence. The results of molecular modeling of the novel and standard triplex structures are consistent with the biochemical and biological data.